Many imaging methods have been developed to take advantage of phase contrast. The most widely used technique with lens-based far-field microscopes is the Zernike method developed by Frits Zernike. Features that are difficult or impossible to observe in absorption contrast can be effectively studied in phase contrast mode, such as biological samples.
The set up for Zernike phase contrast imaging is similar to microscopes for absorption contrast, but a phase plate is used. The phase plate is usually placed at or near the back focal plane of the objective lens to shift the phase of the unscattered beam by π/2 or 3π/2. The unscattered light then interferes with the diffracted light to produce a phase contrast image. This method has been widely used in light microscopy and x-ray microscopy, such as full-field transmission microscopes, with great success.
One drawback of Zernike phase contrast imaging is the mixing of absorption and phase contrast signals and the resulting halo-like artifacts that occur at features' edges. These artifacts can make image interpretation difficult. Thus, generally, this Zernike phase contrast imaging is usually acceptable for observing the features' morphology qualitatively, particularly in two dimensions (2D). With three dimensional (3D) imaging, e.g. computed tomography (CT) techniques, however, these artifacts will lead to severe distortions and amplified artifact structures in the 3D data. This is because the CT algorithm requires each 2D projection image to consist of the linear sum of some characteristic through the sample, e.g. the attenuation coefficient in the case of absorption contrast images. In order to effectively combine the phase-contrast imaging technique with 3D CT imaging, one must derive the linear phase shift through the sample from images that have both absorption and phase contrast signals. Another challenge is the automated separation of specimen constituents by segmentation after the tomographic reconstruction of a tilt series when these artifacts are present.
Recently, quantitative phase reconstruction from differential phase contrast images has been demonstrated in a scanning x-ray microscopy system using a segmented detector system and a Fourier filtering technique. This method inverts the contrast transfer functions of the imaging system similar to a Wiener filter.